1. Field of the Invention
The present invention relates generally to the field of RNA isolation, more specifically it concerns a faster, more efficient method for the isolation of messenger RNA (mRNA) from total RNA.
2. Description of Related Art
In eukaryotic organisms, one trait shared by all mRNAs is the presence on the 3xe2x80x2 end of a stretch of tens to hundreds of adenine (A) residues. This has been used as a means for separating these molecules away from rRNA and other non-mRNA species that lack this xe2x80x9cpolyA tail.xe2x80x9d The standard protocol for the selection of polyA+ RNA is based on the method of Aviv and Leder (1972), wherein a short stretch of DNA consisting solely of thymidine (T) residues (xe2x80x9coligo-dTxe2x80x9d) is affixed to an insoluble matrix. The original procedure used oligo-dT covalently linked to cellulose poured into a column. This is then used as a selective immobilization matrix for mRNA in the sample by setting up conditions that favor formation of RNA-DNA double strands. The total RNA sample is applied to the column in an appropriate salt buffer (originally 0.5 M KCl in 10 mM Tris, pH 7.5), encouraging hybridization to the polyA stretches found solely at the 3xe2x80x2 ends of mRNA. The column is then subjected to extensive washing with the application buffer (containing 0.5 M KCl), then a lower-ionic-strength solution (0.1 M KCl), followed by elution of mRNA with 10 mM Tris (pH 7.5). Subsequent modifications on this original procedure have retained the basic process of hybridization to immobilized oligo-dT in approximately 0.5 M salt, but have changed the format from columns to batch procedures to allow the procedure to be performed faster and have used NaCl or LiCl as the salts.
Further changes have been the replacement of cellulose with plastic or glass beads as the immobilization matrix, some of which are impregnated with ferrous material giving them a magnetic quality. This magnetic quality allows such magnetic beads, as they are referred to, to be batch isolated on magnetic stands rather than requiring gravity or centrifugal force to pellet or filter separate. A further wrinkle in the procedure is the use of a biotin-streptavidin linkage in the connection between oligo-dT and bead, where the oligo is biotinylated and the bead is covalently coupled to streptavidin. The hybridization can be performed in solution with this procedure, linking the oligo-dT-mRNA hybrids to the beads in a subsequent step.
This procedure has tended to be an inefficient method for the separation of rRNA from the mRNA. rRNA carryover levels are often high enough to provide the same problems as presented with total RNA, especially for analysis of rare transcripts. In fact, it usually requires two or more passages through a matrix of choice to produce RNA sufficiently low in rRNA levels to provide a useful sample. This requirement for repetitions of the entire selection procedure can lead to several disadvantageous side effects, which can extend the hours required to perform the procedure. Additionally, the representational distribution of various mRNAs may become altered (or more altered), or the unavoidable losses associated with the repeated procedure may reduce the level of the commonly-sought low-abundance messages beyond the limits of detection. While these drawbacks may be tolerable in a research lab, it a major problem in a diagnostic setting where simplicity, speed and reliability are driving characteristics for a viable assay.
Factors affecting the carryover of rRNA have not been thoroughly studied. Remedial measures have been taken with a vague notion of enhancing the xe2x80x9cgoodxe2x80x9d interaction in the extended A:T hybrid desired by using low ionic-strength (high-stringency) washes and trying to find more xe2x80x9cinertxe2x80x9d materials to use as support. Current procedures use washes at lower ionic strength to remove rRNA that is non-specifically bound to the matrix and the oligo dT. To minimize the non-specific binding of rRNA, novel matrix compositions distinct from cellulose have been pursued, using beads made out of synthetic polymers like latex, polystyrene, other plastics, or even glass. A further modification to enable the streamlining of washing routines was the introduction of plastic or glass beads impregnated with magnetite. This enables the capture of all the beads in a microcentrifuge tube by pulling them to the side with a powerful magnet. Supernatants can be aspirated away to the bottom of the tube, leaving very little carryover of each wash.
Isostabilizing agents, such as tetramethylammonium (TMA+) and tetraethylammonium (TEA+) ions and the amino acid betaine, equalize the hydrogen bonding strength of the G-C and A-T base pairs when used at the appropriate concentrations (Jacobs et al., 1988; Jacobs et al., 1985; Gitschier et al., 1986; Melchior et al., 1973; Rees et al., 1993; Wood et al., 1985; Wozney, 1990). However, while tetramethylammonium chloride (TMAC) and tetraethylammonium chloride (TEAC) have been studied with respect to their effect on thermal transition effects on double-stranded nucleic acid (Golas et al., 1980) and have been employed for hybridization generally (U.S. Pat. No. 5,759,777), they have not been used with a mixed RNA population to facilitate mRNA isolation since some of the problems associated with mRNA isolation with existing techniques were unrecognized.
Thus, there is a need to identify the factors affecting mRNA isolation. With this information, compositions and methods for improved separation of mRNA from other RNA molecules can be developed. Such compositions and methods could facilitate mRNA isolation and purification, for example by increasing the speed of the process or increasing the purity of the end product, which is necessary for scientific research, as well as diagnostic and therapeutic purposes.
The present invention takes advantage of the discovery that some problems with mRNA isolation stems from rRNA carryover that is based not on rRNA interactions with the targetting molecule, oligo-dT, but on rRNA interactions with the targeted molecule, mRNA. While hybridization between oligo-dT and poly(A) RNA is based solely on A:T base-pairing, interactions between rRNA and mRNA involves both A:T and G:C base-pairing. A G:C base pair is stronger than an A:T or A:U base pair, such that the advantage of exact complementation between A and T residues for long stretches (between the targeting molecule and the targeted molecule) are less significant in the face of stronger G:C base pairing between the targeted molecule, mRNA, and contaminating rRNA, even though the stretches may be localized (or shorter) or less exact. Thus, the invention is based on the additional discovery that mRNA isolation is affected by reducing the hybridization between contaminating rRNA and mRNA through the use of an isostabilizing agent, such as TMAC or TEAC salts or the amino acid betaine, which minimizes any difference in bond strength between A:T and G:C basepairs.
The present invention concerns methods, compositions, and kits that facilitate mRNA isolation by increasing the ease or convenience of mRNA isolation, reducing contamination by other non mRNA molecules, increasing the concentration of mRNA, reducing the amount of time to effect mRNA isolation, reducing degradation of RNA in a sample, or any other way that facilitates mRNA isolation.
Thus, in some embodiments of the present invention, methods for purifying mRNA from a sample are contemplated. The term xe2x80x9cpurifyingxe2x80x9d refers to the isolation of mRNA, such that the concentration of mRNA compared to other components increases relative to other components, such as other RNA species. Throughout this application, the terms xe2x80x9cmRNAxe2x80x9d and xe2x80x9cpoly(A) RNAxe2x80x9d are used synonymously since mRNA from most non-bacterial organisms have a poly(A) tail. Any poly(A) RNA from any species is specifically contemplated for isolation using the methods of the present invention. xe2x80x9cPoly(A) RNAxe2x80x9d includes any RNA species with consecutive xe2x80x9cAxe2x80x9d residues toward the 3xe2x80x2 end of an RNA transcript, including those with greater than 10 xe2x80x9cAxe2x80x9d residues.
In some embodiments, poly(A) RNA is isolated from a sample by incubating the sample with an isostabilizing salt and a component that selects the poly(A) RNA as the targeted molecule. The component may be a nucleic acid molecule, RNA or DNA, comprising contiguous xe2x80x9cTxe2x80x9d and/or xe2x80x9cUxe2x80x9d residues (xe2x80x9cpoly(T) or poly(U) nucleic acid moleculexe2x80x9d). In a method of the invention, a composition including the sample, an isostabilizing salt and a poly(T) or poly(U) nucleic acid molecule is incubated under conditions that allow poly(A) RNA to hybridize with the poly(T) or poly(U) nucleic acid molecule. Subsequently, the poly(T) or poly(U) nucleic acid hybridized to mRNA can be isolated or separated from the remainder of the sample. In some embodiments of the invention, it is specifically contemplated that the targeting molecule or poly(T)/poly(U) nucleic acid is not labeled with a compound that allows it to be detected, such as a radioisotope, or calorimetric label/fluorophore, as many embodiments of the invention are directed to isolation and not detection of the targeted molecule with a targeting molecule. The term xe2x80x9cincubatexe2x80x9d refers to maintaining components under certain conditions. The components described herein may also be admixed, mixed, combined, blended, or commingled.
Compositions and methods of the invention involve an isostabilizing agent such as an ion or salt. An isostabilizing agent refers to a solute that affects the hybridization between complementary regions of nucleic acids in such a way that, at certain concentrations of the solute, they negate the difference in the hydrogen bonding between adenine-thymine/uridine (A-T/U) and guanine-cytidine (G-C) base pairs. In some embodiments of the invention tetramethylammonium (TMAC) or tetraethylammonium chloride (TEAC) is specifically contemplated as the isostabilizing agent. In another embodiment betaine is the isostabilizing agent. In still further embodiments, the isostabilizing agent is triethylamine hydrochloride, quinuclidine hydrochloride, or 1,1xe2x80x2-spirobypyrrolidnium bromide. The isostabilizing agent may be added to a sample directly or it may be provided to the sample as a solution containing other components. The isostabilizing agent may be in a binding solution (which facilitates hybridization between the targeted nucleic acid and the component that selects the targeted nucleic acid), wash solution, or elution solution. Other components of the solutions may include buffers, water, detergents, and other salts. In some embodiments, it is specifically contemplated that certain anions, particularly chaotropic anions, are absent from any solution.
In some embodiments of the invention, the concentration of the isostabilizing agent in a composition comprising a sample and a poly(T) nucleic acid is between about 1.0 M and about 3.0 M, between about 1.2 M and about 2.4 M, or between about 1.5 M and about 2.0 M. In some embodiments, the concentration of the isostabilizing agent in a composition comprising a sample that is incubated under hybridization conditions is about 2.0 M. As discussed above, the isostabilizing agent may be provided to the sample as a binding solution; the concentration of the isostabilizing agent in the binding solution will allow for the concentration of the isostabilizing agent in the composition comprising the sample to be in the ranges discussed above. In some embodiments, the concentration of the isostabilizing agent in a binding solution is xe2x80x9cxxe2x80x9d times greater than the concentration needed in the composition comprising the sample. Thus, in an embodiment in which the concentration of the isostabilizing agent in composition comprising the sample is about 2.0 M, a 2xc3x97 binding solution comprises an isostabilizing agent in a concentration of about 4.0 M, a 3xc3x97 binding solution comprises an isostabilizing agent in a concentration of about 6.0 M, etc. Thus, in some embodiments of the invention, a component is provided to the sample in a concentrated form that allows it to be diluted so as to achieve the proper final concentration so as to achieve a desired effect.
A detergent may be added to a composition comprising the sample or it may be comprised in a binding solution that is incubated with the sample. In any of the embodiments of the invention, it is contemplated that a component may be added directly to a composition containing the sample or it may be provided to the sample in a solution. In some embodiments, the detergent is CHAPS or Triton X-100. The final concentration of CHAPS in the composition may be between about 0.5% and about 2.0%, while the final concentration of Triton X-100 in the composition is between about 0.01% and about 0.1%. A final concentration of about 0.017% for the detergent is specifically contemplated, particularly with respect to Triton X-100. A detergent may be included in the binding, wash, or elution solution. In some embodiments, a detergent is specifically contemplated in the binding solution, and thus, it may be absent from the wash or elution solution. Furthermore, while cations other than the isostabilizing agent may be present in the binding, wash, or elution solution, the absence of such cations in such solutions is also specifically contemplated. For example, in some embodiments, Tris is absent in the binding solution.
The sample may be any composition that contains mRNA, including tissue or cell lysate, which refers to a composition of substances from the lysis of cells (or cells from a tissue). Methods of the invention may further comprise steps for preparing a tissue lysate for subsequent mRNA isolation using the method described herein. The use of lysate preparation protocols is specifically contemplated, including the use of guanidinium isothiocyanate for preparing a lysate. Furthermore, in some embodiments, a non-reacting structure such as glass may be employed for lysate preparation, to facilitate the use of methods and compositions for isolating mRNA described herein.
A poly(T) or poly(U) nucleic acid molecule refers to a nucleic acid composed of either RNA or DNA, though a poly d(T) nucleic acid molecule is specifically contemplated. Embodiments discussed with respect to a poly d(T) nucleic acid molecule apply with respect to a poly(T) RNA nucleic acid as well. Such a nucleic acid molecule comprises 1) at least 50% xe2x80x9cTxe2x80x9d and/or xe2x80x9cUxe2x80x9d residues across the entire molecule, though a nucleic acid molecule comprising greater than 70% or 100% xe2x80x9cTxe2x80x9d and/or xe2x80x9cUxe2x80x9d residues is specifically contemplated as part of the invention or 2) comprises a stretch of contiguous xe2x80x9cTxe2x80x9d and/or xe2x80x9cUxe2x80x9d residues of at least 14 nucleobases, though a stretch of at least 25, 50, or 100 contiguous xe2x80x9cTxe2x80x9d and/or xe2x80x9cUxe2x80x9d residues is specifically contemplated. The use of a poly(U) nucleic acid molecule may be used in place of a poly(T) nucleic acid and vice versa. As discussed above, other components instead of a poly(T) or poly(U) nucleic acid molecule may be employed to target a subset of poly(A) RNA molecules or non poly(A) RNA molecules; such components will similarly use hybridization to isolate such molecules.
In some embodiments of the invention, a composition comprising a sample, poly(dT) nucleic acid molecule, and an isostabilizing agent may first be heated at a temperature between about 60xc2x0 C. and about 90xc2x0 C., or between about at least 70xc2x0 C. and about 90xc2x0 C., prior to incubation under hybridization conditions. Temperatures of about 60xc2x0 C., 70xc2x0 C., 80xc2x0 C., and 90xc2x0 C. are specifically contemplated. In some embodiments, hybridization conditions comprise incubating the composition between about 15xc2x0 C. and 50xc2x0 C. for at least 3 minutes to 48 hours, or at least 10 minutes to 48 hours, though longer times are contemplated insofar as substantial RNA degradation does not occur. In additional embodiments, incubation time for hybridization is at least 20 minutes, 1 hour, 4 hours, or 8 hours. During hybridization or binding, the sample may be gently rocked. Furthermore, in some embodiments, the binding solution or a solution containing an isostabilizing agent is discarded and additional solution added to the sample; this may be done multiple times.
Methods of the present invention also include a wash step in some embodiments. Poly(A) RNA may be washed one, two, three, four, five, six, seven, eight, nine, ten, or more times. The wash step may be implemented before or after excess liquid from the sample and/or binding solution is removed. The wash step involves incubating the poly(dT) nucleic acid and poly(A) RNA hybridized to it with a wash solution. In some embodiments, the wash solution contains an isostabilizing agent, such as TMAC or TEAC in a concentration less than its concentration in the composition exposed to hybridization conditions or in the binding solution. The final concentration of the isostabilizing agent during a wash step of a composition comprising poly(A) RNA will be about 0.05 M to about 3.0 M; in some embodiments, the final concentration is below about 0.5 M (low salt wash solution), while in others it is greater than about 2.0 M (high salt wash solution) (medium salt wash solution is between 0.5 M and 2.0 M). A final concentration of the isostabilizing agent during the wash step is specifically contemplated to be 0.4 M. In a specific embodiment the poly(dT) or poly(U) nucleic acid molecule and the hybridized poly(A) RNA are washed at least once in a wash solution with an isostabilizing agent concentration greater than about 1.2 M and at least once in a wash solution with an isostabilizing agent concentration of less than about 0.5 M. In further embodiments, a sample is washed with a low, medium, and/or high salt wash solution. As discuss above, the wash solution may be diluted from a higher concentration, for example, 2xc3x97 (or 2xc3x97) concentration, to a final concentration of 1xc3x97. Thus, for example, in one embodiment of the invention, a 2xc3x97binding solution containing 4 M TMAC and 0.035% Triton X-100 is mixed with an equal volume of sample to achieve a final concentration for hybridization reaction of 2 M TMAC and 0.017% Triton X-100. In many embodiments of the invention, the final concentration of an isostabilizing agent during the binding step is higher than the final concentration of an isostabilizing agent during the wash step.
While the invention is specifically contemplated to omit guanidinium isothiocyanate, a reagent frequently used with RNA isolation, in some embodiments, guanidinium is included in the binding step/solution, wash step/solution, or elution step/solution of the isolation method/compositions. The final concentration of guanidinium in a composition containing poly(A) RNA is between about 0.4 M and about 2.0 M.
A poly(dT) or poly(U) nucleic acid may be a synthetic oligonucleotide. In some embodiments, the oligonucleotide is linked, covalently in some embodiments, or physically attached to a non-reacting structure. In other cases, the oligonucleotide is labeled with a compound that reacts with a second compound physically or covalently linked to a non-reacting structure. A xe2x80x9cnon-reacting structurexe2x80x9d refers to a substance that does not chemically react with the targeted moleculexe2x80x94mRNAxe2x80x94so it can be used to aid in separating the targeted molecule from non-targeted molecules. In some embodiments the non-reacting structure is immobilized. In other embodiments, the non-reacting structure is a bead, cellulose, particulate solid, or other matrix. The bead used may be glass or plastic, and it may be synthetic and/or magnetized. If it is magnetized, the bead may be used in conjunction with a magnetic field. Furthermore, in some embodiments, the non-reacting structure and sample may be centrifuged, filtered, dialyzed, or captured (with a magnet). When the structure is centrifuged it may be pelleted or passed through a centrifugible filter apparatus. The structure may also be filtered, including filtration using a pressure-driven system. Any number of ways of separating or isolating a non-reacting structure may be employed.
In some embodiments, the targeting molecule such as a poly(dT) may be biotinylated or otherwise labeled so as to facilitate isolation. Because biotin reacts with avidin/streptavidin, avidin or streptavidin may be employed in conjunction with a biotinylated substance. The avidin or streptavidin may be linked to a non-reacting structure, such as beads or the surface of a microtiter plate. The non-reacting structure may then be separated from the remainder of the sample to isolate the biotinylated targeting molecule, which itself is bound to the targeted molecule. Thus, in some embodiments, a biotinylated oligo-dT may be employed in conjunction with streptavidin beads to isolate poly(A) RNA molecules. The beads may then be physically separated from the sample to isolate the poly(A) RNA. In some embodiments, a peptide nucleic acid molecule (PNA) may be used as the targeting molecule.
In some embodiments, the method further comprises washing the non-reacting structure before or after exposure to a sample containing RNA, or it may be blocked prior to incubation with a sample by employing a substance that reduces non-specific binding, such as tRNA. Such substances include polymers such as ficoll, polyvinulpyrrolidine, heparin, or dextran sulfate.
Additional embodiments of the methods described herein include a step in which poly(A) RNA is eluted or separated from a non-reacting structure or a molecule (such as streptavidin) linked to the non-reacting structure. The targeted molecule may be eluted with an eluting solution having low ionic strength, which refers to a net concentration of positive or negative charges of less than 0.01 equivalents/L. The eluting solution may include a chelating salt. The eluting solution may have buffers such as Tris and/or EDTA. In some embodiments, the eluting solution includes sodium citrate, which may be in a concentration in the eluting solution of about 0.1 mM to 2 mM, or EDTA-2 Na in a concentration between about 0.1 and 1 mM. Following elution, mRNA may be collected; in some embodiments, the RNA is precipitated using standard salt/alcohol precipitation or it is collected using a column, matrix, or filter for concentrating a nucleic acid.
While many embodiments of the present invention concern mRNA or poly(A) RNA, it is contemplated that the present invention can be implemented with respect to other RNA species (tRNA or rRNA) or a subset of poly(A) RNA. For example, methods and compositions of the invention may be used to separate mRNA from other RNA species in order to purify the other RNA species; that is, mRNA may be isolated away from rRNA, so that the rRNA population is more purified.
Other embodiments of the invention are directed to compositions for achieving purification or isolation of nucleic acid molecules. Kits containing such compositions are specifically contemplated as part of the invention. Compositions and/or apparatuses in the kit would be in a suitable container means, which refers to a container that allows the composition and/or apparatus to be stored and used in methods of the present invention. The container may include synthetic material, or it may include glass material. Kits of the present invention may include one or more of the following:
1) binding solution;
2) wash solution;
3) elution or eluting solution;
4) detergent;
5) poly(dT) oligonucleotide;
6) non-reacting structure;
7) capture device; and
8) RNase inhibitor.
In some embodiments, a poly(dT) oligonucleotide is provided. The poly(dT) oligonucleotide may be linked chemically or physically to a non-reacting structure, which may be cellulose or some other particulate structure. Alternatively, the oligonucleotide may be biotinylated and an avidin/streptavidin-coupled non-reacting structure provided, such as avidin/streptavidin beads. The beads may be plastic, glass, and/or magnetized. If the beads are magnetized, a magnet is included in some embodiments of the invention, such as in the form of a magnetic stand. The kit may contain a binding solution, a wash solution, and/or an elution solution that includes an isostabilizing agent, such as TEAC or TMAC, in various embodiments. Embodiments of these solutions with respect to the methods of the invention pertain to kit embodiments as well. As discussed above, solutions may be provided in a kit in a concentration that is a factor xe2x80x9cxxe2x80x9d greater than the final concentration of components in the solution that are needed to effect purification of poly(A) RNA. In some embodiments of the invention, the solution is a 2xc3x97, 5xc3x97, or 10xc3x97 solution, though in some embodiments, a solution containing an isostabilizing salt, such as the binding solution, is in a concentration of no greater than about 3xc3x97.
Also as discussed above, components in the binding or wash step may be added to a composition containing sample as a solution with other components or it may be added individually, or at least initially separate from an isostabilizing agent, to the composition. For example, a detergent such as Triton X-100 or CHAPS may be included in a binding solution of the kit or it may be provided in the kit simply as a detergent solution that is added to a composition in addition to binding solution. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components, such as 2xc3x97, 5xc3x97, or 10xc3x97. It is specifically contemplated that the concentration of the isostabilizing agent in the binding solution is between about 1.0 M and about 5.0 M, is about 4.0 M, or is about 2.0 M. Also specifically contemplated is a wash solution with an isostabilizing agent concentration of between about 0.1 M and 2.0 M. In some embodiments of the invention, the binding or wash solution may include guanidinium isothiocyanate, though in some embodiments this chemical is specifically contemplated to be absent. The concentration of guanidinium may be between about 0.4 M and about 3.0 M
In some embodiments of the invention, an elution solution is provided. The elution solution may constitute water or TE, or it may contain sodium citrate. An example of a TE solution is 10 MM TrisCl and 1 mM EDTA, pH 7.5. The solution may have a low ionic strength. In additional embodiments, sodium citrate is included in an elution solution or is included in the kit separate from an isostabilizing agent. The concentration of the salt in the elution solution is, in some embodiments, between about 0.1 mM and about 100 mM.
In so me embodiments, the concentration of the detergent in the binding solution is between about 0.001% to about 1.0%. Alternatively, detergent may be provided individually in a solution at a concentration of between about 0.001% and about 10.0%. Any solution of the kit may further comprise a buffer such as Tris, phosphate, or any xe2x80x9cGoodxe2x80x9d buffer (Ferguson et al., 1980, specifically incorporated by reference, describing such buffers).
Other embodiments of the invention include kits containing devices or apparatus for physically separating the targeted molecule from the remainder of the sample (xe2x80x9ccapture devicesxe2x80x9d). A magnetic stand is a capture device of the present invention. Other embodiments include centrifugation as a capture means, as well as a filtration device useable with either differential air pressure or centrifugal force. Other devices that may be used are a microtiter plate format with oligo-dT directly attached to the plate or attached through a biotin-streptavidin/avidin linkage.
Specifically contemplated is a kit containing a poly(dT) oligonucleotide linked to cellulose; hybridization solution comprising tetramethylammonium (TMAC) in a concentration of between about 2 M and 4 M and Triton X-100 in a concentration of between about 0.01% and about 0.1%; wash solution comprising TMAC in a concentration of about 0.4 M; and elution solution comprising sodium citrate in a concentration of between about 1 mM and about 2 mM.
Also specifically contemplated is a kit containing: a poly (dT) oligonucleotide linked to beads, of either a magnetic or nonmagnetic nature; hybridization solution comprising TMAC in a concentration of between about 2 M and 4 M and Triton X-100 in a concentration of between about 0.01% and about 0.1%; wash solution comprising TMAC in a concentration of about 0.4 M, and an elution solution comprising sodium citrate in a concentration of between about 1 mM and about 2 mM.
The use of the word xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d when used in conjunction with the term xe2x80x9ccomprisingxe2x80x9d in the claims and/or the specification may mean xe2x80x9cone,xe2x80x9d but it is also consistent with the meaning of xe2x80x9cone or more,xe2x80x9d xe2x80x9cat least one,xe2x80x9d and xe2x80x9cone or more than one.xe2x80x9d
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.